Mature Rat Research Articles

The life, death and regenerative ability of immature and mature rat retinal ganglion cells are influenced by their birthdate

The extensive period of retinal ganglion cell (RGC) neurogenesis in the rat is associated with a protracted sequence of arrival of their axons into central targets such as the superior colliculus (SC) (Dallimore et al., 2002). Using in utero 5-bromo-2'-deoxyrudine (BrdU) injections to label early (embryonic day (E) 15) or late (E18 or E19) born RGCs, we now show that E15 RGCs with axons that enter the SC prenatally undergo programmed cell death earlier than late-born RGCs whose axons only reach the SC late in the first postnatal week. These late-born RGCs do not begin to die until postnatal day (P) 5/6. Removal of retrograde trophic support by P1 SC ablation initially only affects E15 RGCs; however by P5 death of late-born RGCs is increased, confirming that a switch to target dependency is delayed in this cohort. In a further experiment it was found that, following complete rostral SC transection at P2, the proportion of post-lesion axons originating from E19 RGCs was significantly greater than the proportion that normally makes up the retinotectal projection. Thus, even in neonatal brain, uninjured late-arriving axons are more likely to grow across a lesion site than injured axons undergoing regeneration. To study if birth date also affects regenerative potential in adulthood, autologous peripheral nerve (PN) was grafted onto the cut optic nerve in mature BrdU labelled rats. We found that, compared to E15 RGCs, a significantly greater proportion of late-born RGCs survived axotomy, but comparatively fewer of these surviving E19 RGCs regrew an axon into a graft. In summary, this research shows that the birthdate of RGCs significantly impacts on their subsequent life history and response to injury. Understanding how developing central nervous system (CNS) neurons acquire dependency on target-derived trophic support may lead to new strategies for enhancing survival and regeneration in adult CNS.

Insulin resistance syndrome blunts the mitochondrial anabolic response following resistance exercise

Metabolic risk factors associated with insulin resistance syndrome may attenuate augmentations in skeletal muscle protein anabolism following contractile activity. The purpose of this study was to investigate whether or not the anabolic response, as defined by an increase in cumulative fractional protein synthesis rates (24-h FSR) following resistance exercise (RE), is blunted in skeletal muscle of a well-established rodent model of insulin resistance syndrome. Four-month-old lean (Fa/?) and obese (fa/fa) Zucker rats engaged in four lower body RE sessions over 8 days, with the last bout occurring 16 h prior to muscle harvest. A priming dose of deuterium oxide ((2)H(2)O) and (2)H(2)O-enriched drinking water were administered 24 h prior to euthanization for assessment of cumulative FSR. Fractional synthesis rates of mixed (-5%), mitochondrial (-1%), and cytosolic (+15%), but not myofibrillar, proteins (-16%, P = 0.012) were normal or elevated in gastrocnemius muscle of unexercised obese rats. No statistical differences were found in the anabolic response of cytosolic and myofibrillar subfractions between phenotypes, but obese rats were not able to augment 24-h FSR of mitochondria to the same extent as lean rats following RE (+14% vs. +28%, respectively). We conclude that the mature obese Zucker rat exhibits a mild, myofibrillar-specific suppression in basal FSR and a blunted mitochondrial response to contractile activity in mixed gastrocnemius muscle. These findings underscore the importance of assessing synthesis rates of specific myocellular subfractions to fully elucidate perturbations in basal protein turnover rates and differential adaptations to exercise stimuli in metabolic disease.

Activity‐ and age‐dependent modulation of GABAergic neurotransmission by System A‐mediated glutamine uptake

GABAergic neurotransmission adapts to maintain normal brain function in a wide range of activity states through multiple mechanisms; pre-synaptic control of quantal size has only recently gained recognition as one of those mechanisms. GABA synthesis from glutamate is coupled with vesicular packaging, and therefore the supply of glutamate can affect inhibitory synaptic strength. Because System A transporters supply glutamine to neurons, where it is converted to glutamate, we hypothesized that regulation of the activity of these transporters could alter glutamine uptake and provide a mechanism to link supply to demand for neurotransmitter GABA. In immature and mature rat hippocampus, after a period of hyperexcitability, we observed a System A-dependent enhancement of inhibitory synaptic strength along with an increase in System A activity in synaptosomes under the same conditions. Under resting conditions, System A's contribution of glutamine to synaptic GABA diminished with age, correlating with reduced SNAT1/SAT1 expression and, even more so, with its activity on synaptic membranes. We conclude that System A activity is highly regulated, by depolarization and developmental cues, to dynamically modulate GABAergic transmission. Our evidence suggests that SNAT1/SAT1 is the transporter that plays a critical role in dynamically modulating inhibition in response to metabolic demands.

Regulation of CCN2/Connective tissue growth factor expression in the nucleus pulposus of the intervertebral disc: Role of Smad and activator protein 1 signaling

To investigate transforming growth factor beta (TGFbeta) regulation of connective tissue growth factor (CTGF) expression in cells of the nucleus pulposus of rats, mice, and humans. Real-time reverse transcription-polymerase chain reaction and Western blot analyses were used to measure CTGF expression in the nucleus pulposus. Transfections were used to measure the effects of Smads 2, 3, and 7 and activator protein 1 (AP-1) on TGFbeta-mediated CTGF promoter activity. CTGF expression was lower in neonatal rat discs than in skeletally mature rat discs. An increase in CTGF expression and promoter activity was observed in rat nucleus pulposus cells after TGFbeta treatment. Deletion analysis indicated that promoter constructs lacking Smad and AP-1 motifs were unresponsive to treatment. Analysis showed that full-length Smad3 and the Smad3 MH-2 domain alone increased CTGF activity. Further evidence of Smad3 and AP-1 involvement was seen when DN-Smad3, SiRNA-Smad3, Smad7, and DN-AP-1 suppressed TGFbeta-mediated activation of the CTGF promoter. When either Smad3 or AP-1 sites were mutated, CTGF promoter induction by TGFbeta was suppressed. We also observed a decrease in the expression of CTGF in discs from Smad3-null mice as compared with those from wild-type mice. Analysis of human nucleus pulposus samples indicated a trend toward increasing CTGF and TGFbeta expression in the degenerated state. TGFbeta, through Smad3 and AP-1, serves as a positive regulator of CTGF expression in the nucleus pulposus. We propose that CTGF is a part of the limited reparative response of the degenerated disc.

Possible involvement of DNA methylation in NKCC1 gene expression during postnatal development and in response to ischemia

In CNS, GABA(A) receptor-mediated responses switch from depolarization to hyperpolarization during postnatal development. This switch is mediated by developmental down-regulation of inwardly directed Na(+)-K(+)-2Cl(-) co-transporter type 1 (NKCC1) and up-regulation of outwardly directed K(+)-Cl(-) co-transporter type 2. While several factors have been shown to regulate K(+)-Cl(-) co-transporter type 2 expression, little is known about the mechanisms by which the expression of NKCC1 is regulated during postnatal development. Here, we report a novel epigenetic mechanism underlying the developmental regulation of NKCC1 gene expression in the rat cerebral cortex. In vitro DNA methylation of the NKCC1 promoter region, which contains a high density of cytosine-phosphodiester-guanine islands, significantly decreased the expression of NKCC1 mRNA, and the degree of methylation of the NKCC1 promoter region significantly increased during postnatal development. In addition, treatment with 5-aza-2'-deoxycytidine, a specific DNA methyltransferase inhibitor, elicited an increase in the expression of NKCC1 mRNA and protein in cortical slice cultures. Focal ischemic injury induced by the occlusion of the middle cerebral artery led to the re-expression of NKCC1 mRNA and protein even in the mature rat cortex. The re-expression of NKCC1 mRNA and protein in the injured cerebral cortex was related to a decrease in the methylation status of the NKCC1 promoter region. Our results indicate that epigenetic mechanisms, such as DNA methylation, might be involved in the regulation of NKCC1 gene expression during postnatal development as well as under pathological conditions.

The K+–Cl−cotransporter KCC2 promotes GABAergic excitation in the mature rat hippocampus

GABAergic excitatory [K(+)](o) transients can be readily evoked in the mature rat hippocampus by intense activation of GABA(A) receptors (GABA(A)Rs). Here we show that these [K(+)](o) responses induced by high-frequency stimulation or GABA(A) agonist application are generated by the neuronal K(+)-Cl() cotransporter KCC2 and that the transporter-mediated KCl extrusion is critically dependent on the bicarbonate-driven accumulation of Cl() in pyramidal neurons. The mechanism underlying GABAergic [K(+)](o) transients was studied in CA1 stratum pyramidale using intracellular sharp microelectrodes and extracellular ion-sensitive microelectrodes. The evoked [K(+)](o) transients, as well as the associated afterdischarges, were strongly suppressed by 0.5-1 mm furosemide, a KCl cotransport inhibitor. Importantly, the GABA(A)R-mediated intrapyramidal accumulation of Cl(), as measured by monitoring the reversal potential of fused IPSPs, was unaffected by the drug. It was further confirmed that the reduction in the [K(+)](o) transients was not due to effects of furosemide on the Na(+)-dependent K(+)-Cl() cotransporter NKCC1 or on intraneuronal carbonic anhydrase activity. Blocking potassium channels by Ba(2+) enhanced [K(+)](o) transients whereas pyramidal cell depolarizations were attenuated in further agreement with a lack of contribution by channel-mediated K(+) efflux. The key role of the GABA(A)R channel-mediated anion fluxes in the generation of the [K(+)](o) transients was examined in experiments where bicarbonate was replaced with formate. This anion substitution had no significant effect on the rate of Cl() accumulation, [K(+)](o) response or afterdischarges. Our findings reveal a novel excitatory mode of action of KCC2 that can have substantial implications for the role of GABAergic transmission during ictal epileptiform activity.

Three-dimensional finite element modeling of pericellular matrix and cell mechanics in the nucleus pulposus of the intervertebral disk based on in situ morphology

Nucleus pulposus (NP) cells of the intervertebral disk (IVD) have unique morphological characteristics and biologic responses to mechanical stimuli that may regulate maintenance and health of the IVD. NP cells reside as single cell, paired or multiple cells in a contiguous pericellular matrix (PCM), whose structure and properties may significantly influence cell and extracellular matrix mechanics. In this study, a computational model was developed to predict the stress-strain, fluid pressure and flow fields for cells and their surrounding PCM in the NP using three-dimensional (3D) finite element models based on the in situ morphology of cell-PCM regions of the mature rat NP, measured using confocal microscopy. Three-dimensional geometries of the extracellular matrix and representative cell-matrix units were used to construct 3D finite element models of the structures as isotropic and biphasic materials. In response to compressive strain of the extracellular matrix, NP cells and PCM regions were predicted to experience volumetric strains that were 1.9-3.7 and 1.4-2.1 times greater than the extracellular matrix, respectively. Volumetric and deviatoric strain concentrations were generally found at the cell/PCM interface, while von Mises stress concentrations were associated with the PCM/extracellular matrix interface. Cell-matrix units containing greater cell numbers were associated with higher peak cell strains and lower rates of fluid pressurization upon loading. These studies provide new model predictions for micromechanics of NP cells that can contribute to an understanding of mechanotransduction in the IVD and its changes with aging and degeneration.

The Effect of Aging on the Mature Albino Rat Testis, A morphometric Study

The Effect of Aging on the Mature Albino Rat Testis, A morphometric Study

In P14 rat middle cerebral arteries relations between myogenic tone and cytosolic Ca++ are enhanced by myofilament Ca++ sensitization and increased Ca++ influx, but attenuated by high proportions of non‐contractile smooth muscle cells.

To test the hypothesis that relations between myogenic tone and cytosolic Ca++ are enhanced by myofilament Ca++ sensitization but attenuated by high proportions of non‐contractile smooth muscle cells, in immature compared to mature rat cerebral arteries, three protocols were performed in middle cerebral arteries (MCA) from P14 and adult rats. Myofilament Ca2+ sensitivity in β‐escin permeabilized arteries increased with pressure in P14, but not adult, MCA. Cyclopiazonic acid (a release inhibitor) increased diameter and reduced Ca++ in adult MCA, but increased diameter with no change in Ca++ in the P14 MCA. La3+ (an influx inhibitor) increased diameter and decreased Ca++ in adult MCA, but in P14 MCA diameter increased without any significant decline in Ca++. With La3+ and CPA, diameter was passive in adult and P14 MCA, but Ca++ declined only in adult MCA. Colocalization between α‐actin and SM2 myosin was >2‐fold greater in adult than in pup MCA.. These data suggest that compared to adult MCA, pup MCA rely more on myofilament Ca++ sensitization and Ca++ influx to maintain myogenic reactivity. The inability of La3+ to reduce cytosolic Ca++ in the pup MCA appears due to La3+‐insensitive non‐contractile smooth muscle cells that contribute to the spatially averaged measurements of Ca++ but not contraction. Supported by USPHS grant HL54120, HD31266, and HL64867.

Effects of exercise training on dendritic morphology in the cardiorespiratory and locomotor centers of the mature rat brain

It has been shown that dendritic branching in neural cardiorespiratory and locomotor centers can be attenuated with exercise training (ET) initiated immediately after weaning. The purpose of this study was to determine whether neuroplastic changes occur within cardiorespiratory and locomotor centers due to ET after maturation. Male Sprague-Dawley rats (21 days old, n=28) were individually housed in standard cages. At 91 days of age, animals were divided into two groups: untrained (UN; n=14) and trained (TR; n=14). The TR group exercised spontaneously for 50 days on running wheels. ET indexes were obtained, including maximal O2 consumption, percent body fat, resting heart rate, and heart weight-to-body weight ratios. The brain was processed with a modified Golgi-Cox procedure. Impregnated neurons from the periaqueductal gray (PAG), posterior hypothalamic area (PH), nucleus of the tractus solitarius (NTS), cuneiform nucleus (CnF), rostral ventrolateral medulla, nucleus cuneatus, and cerebral cortex were examined. Neurons were traced and analyzed using the Sholl concentric ring analysis of dendritic branching. The mean total number of dendritic intersections with the concentric rings per neuron per animal were compared between UN and TR groups. There were significant differences between UN and TR groups in the PH, PAG, CnF, and NTS in the total number of intersections per animal. In some areas, the effect size was smaller when ET was initiated in mature animals, possibly related to their relatively reduced activity levels. In conclusion, the adult rat brain remains dynamic and adapts to chronic ET. However, some brain areas appear to be more affected if ET is initiated in early postnatal development.

Exogenous thyroid hormone affects myoepithelium and proliferation in the developing rat parotid gland

In the mature rat parotid gland, myoepithelial cells (MEC) invest intercalated ducts, but not acini. During postnatal development, however, these cells differentiate around both intercalated ducts and acini, then translocate to only intercalated ducts during weaning. Previously, we found that thyroxine (T4) accelerates translocation of cells with small secretory granules from acini into intercalated ducts and the number of apoptotic cells increased tremendously with high doses. We present here additional analysis of the effects of T4 on developing rat parotid gland, namely, the distribution of MEC and the proliferation of parenchymal cells. Beginning at age four days, pups were given daily subcutaneous injections of low, medium, and high doses of T4 or vehicle or no injection. At ages 4, 7, 10, and 15 days, glands were excised and processed for light microscopy. Sections were double-immunostained with antibodies against proliferating cell nuclear antigen (PCNA) and actin, and counterstained with hematoxylin. Proliferative activity was assessed via PCNA histochemistry and MEC were identified using actin histochemistry. MEC in the T4 groups invested mostly acini at 15 days in vehicle/normal glands and mostly intercalated ducts after 10 days in the T4 groups. The proliferative activity of acinar cells and MEC in vehicle/normal glands declined progressively with age and T4 increased the rate of this decline in the MEC in a dose-dependent manner. We conclude that T4 accelerates the translocation of MEC from acini to intercalated ducts and that an important mechanism is the more rapid decline in the proliferative activity of MEC than in acinar cells in the T4 groups. Some of the decline in the proliferative activity of all cells in the high and medium dose T4 groups after seven days may have been due to dose-related thyroxine toxicity.

SOCS3 Deletion Promotes Optic Nerve Regeneration In Vivo

Axon regeneration failure accounts for permanent functional deficits following CNS injury in adult mammals. However, the underlying mechanisms remain elusive. In analyzing axon regeneration in different mutant mouse lines, we discovered that deletion of suppressor of cytokine signaling 3 (SOCS3) in adult retinal ganglion cells (RGCs) promotes robust regeneration of injured optic nerve axons. This regeneration-promoting effect is efficiently blocked in SOCS3-gp130 double-knockout mice, suggesting that SOCS3 deletion promotes axon regeneration via a gp130-dependent pathway. Consistently, a transient upregulation of ciliary neurotrophic factor (CNTF) was observed within the retina following optic nerve injury. Intravitreal application of CNTF further enhances axon regeneration from SOCS3-deleted RGCs. Together, our results suggest that compromised responsiveness to injury-induced growth factors in mature neurons contributes significantly to regeneration failure. Thus, developing strategies to modulate negative signaling regulators may be an efficient strategy of promoting axon regeneration after CNS injury.

A comparative study of the gross structure and distribution of spermatogonia-like cells in the testis of pre-pubertal and mature adult rats

The testis of pre-pubertal and those of mature adult albino rats were physically divided into three segments with a view to studying histologically the gross structure and distribution of spermatogonia-derived cells (spermatogonia,spermatocytes, spermatids and mature spermatozoa) in the seminiferous tubules in these segments. After using 50% Mezo stain (organic), spermatogonia-derived cells were manually counted on slide-view. A mixed population of dense uninucleated single or paired type A-like spermatogonia was clearly identifiable in both upper and lower segments as well as in the middle segment of the testis of pre-pubertal rats. In the mature adult rat testis, a mixed population of spermatocytes, oval spermatids and mature spermatozoa, in addition to a mixed population of dense uninucleated and multinucleated type A-like spermatogoniawere identified in seminiferous tubules of all three segments. However, there were larger numbers of clump-forming spermatogonia-like cells in the middle segment of the testis than in other segments in mature adult rats. A ratio of 2:1:1 for middle, upper and lower segments respectively was manually determined. These results suggest that the middle segment of the mature adult albino rat testis contains a highly enriched population of both single and multinucleated A-type spermatogonia-like cells and may be most suitable site for the harvesting of these cells. Key words: Rat, pre-pubertal, testis, spermatogonia, spermatogenesis, mezo stain.

Diverse effects of bacterial cell wall components on mast cell degranulation, cysteinyl leukotriene generation and migration

Nowadays there is more and more evidence that mast cells take part in antibacterial defence. Mast cells have the ability to kill bacteria via phagocytose-dependent or phagocytose-independent ways and express antimicrobial peptides that can directly kill pathogens at their site of entry. What is more, mast cells are capable of processing bacterial antigens for presentation through class I and II MHC molecules. Some data indicate that these cells can release various proinflammatory mediators in response to activation with bacteria and/or their products, however this information is still far from complete. Therefore, in this study we examined the ability of PGN from Staphylococcus aureus, LPS from Eschericha coli and LAM from Mycobacterium smegmatis to stimulate mature rat mast cell degranulation as well as cysteinyl LT generation. We also studied the influence of these bacterial components on mast cell migration. We found that PGN, LPS and LAM all failed to induce mast cell degranulation and histamine release. At the same time, activation of mast cells with these bacterial antigens resulted in generation and release of significant amounts of LT. Moreover, we documented that, even in the presence of laminin, none of the bacterial antigens used stimulated mast cell migration. However, PGN did induce migration of RANTES-primed mast cells, and LPS did stimulate mast cell migratory response after priming with IL-6. Our results show that PGN, LPS and LAM might be among the important bacterial antigens involved in mast cell activation during bacterial infection.

Insulin-mediated Regulation of Decidual Protein Induced by Progesterone (DEPP) in Adipose Tissue and Liver

We analyzed the profile of the genes expressed in human adipose tissue and identified the fat-derived molecules, adiponectin and aquaporin 7, which modulate glucose and lipid metabolism. The same Bodymap analysis revealed abundant expression of the decidual protein induced by progesterone (DEPP) in the white adipose tissue. Northern blot analysis confirmed that human DEPP mRNA was highly expressed in white adipose tissue. Mouse DEPP mRNA was detected in heart, lung, skeletal muscle, and white adipose tissue under feeding state. In contrast, under fasting state, mouse DEPP mRNA was enhanced in lung, skeletal muscle, and white adipose tissue and it appeared also in the liver and kidney, suggesting up regulation of DEPP by fasting. Because fasting-induced DEPP expression was observed in insulin-sensitive organs, we investigated the regulation of DEPP in white adipose tissue and liver. During adipogenesis of mouse 3T3-L1 cells, DEPP mRNA increased in a differentiation-dependent manner similar to adiponectin and aquaporin 7. Treatment of cultured 3T3-L1 mature adipocytes, rat H4IIE, and human HepG2 hepatoma cells with insulin significantly decreased DEPP mRNA levels in dose- and time-dependent manners. IN VIVO experiments showed significant decrease of hepatic and adipose DEPP mRNA levels in refed mice, compared to fasted animals, and also showed significant increase in DEPP mRNA in streptozotocin-induced insulin-deficient diabetic mice. These results indicate that DEPP is a novel insulin-regulatory molecule expressed abundantly in insulin-sensitive tissues including white adipose tissue and liver.

Effects of permethrin, cypermethrin and 3-phenoxybenzoic acid on rat sperm motility in vitro evaluated with computer-assisted sperm analysis

Pyrethroid pesticides, produced and used worldwide, have been reported to impair male reproductive function by reducing sperm count and sperm motility. They are divided into two types: type I pyrethroids including permethrin, etc. and type II pyrethroids including cypermethrin, fenvalerate, cyfluthrin, etc. Our previous study showed that fenvalerate and cypermethrin could reduce sperm motility in vitro. However, it is not clear whether permethrin and 3-phenoxybenzoic acid (3-PBA, the major metabolite of pyrethroids) affect sperm motility directly or indirectly by affecting spermatogenesis via interaction with androgens and/or their receptors. In this study, rat sperm suspensions were treated respectively with permethrin, cypermethrin and 3-PBA, at various concentrations (0, 1, 4, 16, or 64 mmol/L) for various times (1, 2, or 4 h). The motility parameters of sperm were analyzed with a computer-assisted sperm analysis (CASA) system. The differential effects of permethrin and cypermethrin on sperm motility patterns in vitro were also compared. Our study revealed that permethrin and cypermethrin could reduce sperm motility in vitro in a concentration- and time-dependent manner. Marked differences between the two pyrethroids were not found in this study. Moreover, 3-PBA did not reduce sperm motility directly at all concentrations and treatment periods. These results provide further evidence that permethrin and cypermethrin can directly affect mature rat sperm motility.

Mst3b, an Ste20-like kinase, regulates axon regeneration in mature CNS and PNS pathways

Mammalian sterile 20-like kinase-3b (Mst3b/Stk24) regulates axon outgrowth in embryonic cortical neurons in culture, but its role in vivo and in neural repair is unknown. Here we show that Mst3b mediates the axon-promoting effects of trophic factors in mature rat retinal ganglion cells (RGCs) and dorsal root ganglion (DRG) neurons, and is essential for axon regeneration in vivo. Reducing Mst3b levels using shRNA prevented RGCs and DRG neurons from regenerating axons in response to growth factors in culture, as did expression of a kinase-dead Mst3b mutant. Conversely, expression of constitutively active Mst3b enabled both types of neurons to extend axons without growth factors. In vivo, RGCs lacking Mst3b failed to regenerate injured axons when stimulated by intraocular inflammation. DRG neurons regenerating axons in vivo showed elevated Mst3b activity, and reducing Mst3b expression attenuated regeneration and p42/44 MAPK activation. Thus, Mst3b regulates axon regeneration in both CNS and PNS neurons.

The dual dopamine‐glutamate phenotype of growing mesencephalic neurons regresses in mature rat brain

Coexpression of tyrosine hydroxylase (TH) and vesicular glutamate transporter 2 (VGLUT2) mRNAs in the ventral tegmental area (VTA) and colocalization of these proteins in axon terminals of the nucleus accumbens (nAcb) have recently been demonstrated in immature (15-day-old) rat. After neonatal 6-hydroxydopamine (6-OHDA) lesion, the proportion of VTA neurons expressing both mRNAs and of nAcb terminals displaying the two proteins was enhanced. To determine the fate of this dual phenotype in adults, double in situ hybridization and dual immunolabeling for TH and VGLUT2 were performed in 90-day-old rats subjected or not to the neonatal 6-OHDA lesion. Very few neurons expressed both mRNAs in the VTA and substantia nigra (SN) of P90 rats, even after neonatal 6-OHDA. Dually immunolabeled terminals were no longer found in the nAcb of normal P90 rats and were exceedingly rare in the nAcb of 6-OHDA-lesioned rats, although they had represented 28% and 37% of all TH terminals at P15. Similarly, 17% of all TH terminals in normal neostriatum and 46% in the dopamine neoinnervation of SN in 6-OHDA-lesioned rats were also immunoreactive for VGLUT2 at P15, but none at P90. In these three regions, all dually labeled terminals made synapse, in contradistinction to those immunolabeled for only TH or VGLUT2 at P15. These results suggest a regression of the VGLUT2 phenotype of dopamine neurons with age, following normal development, lesion, or sprouting after injury, and a role for glutamate in the establishment of synapses by these neurons.

Opposite effect of membrane raft perturbation on transport activity of KCC2 and NKCC1

In the majority of neurons, the intracellular Cl(-) concentration is set by the activity of the Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) and the K(+)-Cl(-) cotransporter (KCC2). Here, we investigated the cotransporters' functional dependence on membrane rafts. In the mature rat brain, NKCC1 was mainly insoluble in Brij 58 and co-distributed with the membrane raft marker flotillin-1 in sucrose density flotation experiments. In contrast, KCC2 was found in the insoluble fraction as well as in the soluble fraction, where it co-distributed with the non-raft marker transferrin receptor. Both KCC2 populations displayed a mature glycosylation pattern. Disrupting membrane rafts with methyl-beta-cyclodextrin (MbetaCD) increased the solubility of KCC2, yet had no effect on NKCC1. In human embryonic kidney-293 cells, KCC2 was strongly activated by a combined treatment with MbetaCD and sphingomyelinase, while NKCC1 was inhibited. These data indicate that membrane rafts render KCC2 inactive and NKCC1 active. In agreement with this, inactive KCC2 of the perinatal rat brainstem largely partitioned into membrane rafts. In addition, the exposure of the transporters to MbetaCD and sphingomyelinase showed that the two transporters differentially interact with the membrane rafts. Taken together, membrane raft association appears to represent a mechanism for co-ordinated regulation of chloride transporter function.

Short-Term Effects of Parathyroid Hormone on Rat Lumbar Vertebrae

This study is an experimental study in the rat osteopenia model. The aim of this study was to evaluate the short-term effects of daily application of parathyroid hormone (PTH) on bone quality and quantity using a new biomechanical compression test for intact rat lumbar vertebrae. Because of their high clinical relevance, trabecular content and thick cortical shell vertebrae are of high interest for osteoporosis research. Biomechanical stability depends on both trabecular and cortical bone. Anabolic effects on bone after long-term application of PTH have already been proven. After an intraindividual comparison (n = 20), the capability of a new test to identify biomechanical properties of the mature rat model was assessed. In the following, 33 three-month-old rats were ovariectomized. After 10 weeks, the animals were divided into 3 groups. The control group (C) received no additional food supplementation. The other groups received hormone treatment with either estradiol (E) or PTH for another 5 weeks. The effects on bone biomechanical properties and bone microstructure were analyzed. After establishing the new biomechanical test for intact rat lumbar vertebrae, PTH-treated (yield stress: 2.95 N/mm, elastic limit: 2.39 N/mm) and then E-treated (yield stress: 2.13 N/mm, elastic limit: 1.68 N/mm) animals showed superior biomechanical results. Compression strength was significantly improved in these rats in comparison to the control group rats (yield stress: 1.86 N/mm, elastic limit: 1.38 N/mm). In the microradiographic evaluation, PTH significantly improved the morphologic results to produce thicker trabeculae. E led to a more densely branched trabecular network, which was not as important as trabecular thickness for bone stability. After a short-term application, PTH is superior to E in recreating bone biomechanical propertiesand lumbar vertebral microstructure in advanced osteoporosis. The cortical shell and trabecular thickness are primarily responsible for the biomechanical strength of vertebrae.